For many diseases with known genetic causes, gene therapy appears to be the most promising approach to treatment. Most frequently, transgenes are delivered with viral vectors, which often cannot accommodate the entire genomic locus with all of its regulatory sequences and alternative splicing sites. These vectors randomly integrate the exogenous genetic material, possibly leading to insertional mutagenesis. To address these critical limitations, we propose to use human artificial chromosomes (HAC) as vectors for gene therapy studies. HAC can complement gene deficiencies in cultured cells, place no limitation on transgene size, pose no risk of insertional mutagenesis, are highly stable, and deliver prolonged gene expression. In this project, we aim to establish HAC as non-integrating, high capacity vectors for gene therapy in human fibroblast and keratinocyte cell, focusing as a model disease on Xeroderma pigmentosum (XP) in its most common form, XPC. XP, a genetic disorder characterized by development of skin cancers and progressive neurological disturbances, is caused by mutations in the nucleotide excision DNA repair pathway. The XPC protein is a damage-sensing and DNA-binding factor. We expect to demonstrate that cells from XP patients can be protected from UV damage by gene transfer of an intact XPC gene along with its regulatory sequences. To complement the deficiency (Aim 1), we will introduce into patient-derived fibroblasts and keratynocytes an HAC vector containing the entire XPC gene. Secondly, we will generate a functional 3D skin equivalent model for XP using the corrected fibroblasts and keratinocytes and assess whether complementing the XP deficiency restores normal DNA repair (Aim 2). Our research team includes experts who pioneered many of the methods we will use. Zoia Monaco's lab was the first to demonstrate HAC-based complementation of a genetic deficiency in human cells. Daniela Moralli developed an HSV-1 based technique that allows direct generation of HAC in a variety of cell types and was instrumental in adapting the technique to human embryonic stem cells. Jonathan Garlick is a leader in the bioengineering of 3D human skin-like tissues to study stem cell differentiation and cancer, and to provide tissue platforms for drug discovery. Ken Kraemer is an internationally renowned XP expert. Successful completion of these aims will demonstrate the feasibility of HAC-based gene therapy vectors, delivering entire genomic loci into human cells for complementation of genetic deficiencies.